Regulators of G protein Signaling (RGS) proteins are a family of G protein-coupled receptor (GPCR) accessory proteins that are essential for the temporal and spatial control of cell signaling, including signaling downstream of the mu-opioid receptor (MOR). RGS proteins are GTPase accelerating proteins (GAPs) that accelerate the hydrolysis of Galpha bound GTP and promote the formation of inactive Galpha GDP to switch off signaling by GPCRs. We have shown that RGS proteins serve to terminate signaling of MOR to adenylate cyclase and the MAP kinase pathway. On the other hand, efficient signaling of MOR to release intracellular calcium requires RGS protein GAP activity. Thus, RGS activity controls the balance of signaling pathways within a single cell. We recently used a novel tool to explore regulation of GPCR signaling by RGS proteins: a transgenic knock-in mouse that expresses Galpha proteins that are insensitive to the GAP activity of RGS proteins. In these mice antinociception is dependent on the opioid agonist and pain assay employed. For example, in the hot-plate assay loss of RGS regulation potentiates morphine, without affecting methadone, antinociception. In contrast, in the tail-withdrawal assay removal of RGS activity decreases both morphine and methadone antinociception. This suggests the different neuronal pathways involved in these two behaviors show differential sensitivity to RGS protein action. We propose to continue our exploration of these mice to tackle a series of fundamental questions concerning MOR signaling and its relationship to antinociception. For example: Are the differences between antinociceptive tests due to site-specific variation in RGS action? What is the basis of the observed agonist differences? Can the differences be explained by effects at the level of cell signaling? What is the role of other neurotransmitter systems that also use Galpha proteins? Are more clinically-related pain models also regulated by RGS proteins? Our overall conceptual framework is as follows: 1) RGS activity controls the balance of GPCR signaling to multiple pathways and this balance may be disrupted in pain and 2) RGS-induced changes in MOR-mediated-antinociception may reflect an alteration in the balance between neurotransmitter systems, particularly the nociceptin (NOP) system. The proposed studies will advance our understanding of opioid signaling pathways and their regulation by RGS proteins and explain these actions in the context of altered behaviors. Results from this study may be exploited to develop better analgesic drugs.